Methods for selectively capturing and amplifying exons or targeted genomic regions from biological samples

Abstract

In one aspect, the present invention relates to a method for selectively capturing and/or amplifying exons or targeted genomic regions from biological samples. In one embodiment, the method includes the steps of obtaining DNA templates for the targeted genomic region, cloning the DNA templates into cloning vectors to form template DNA clones, constructing libraries of the template DNA clones that cover at least the targeted genomic regions, generating hybridization probes from the DNA template clones in the libraries, capturing the targeted genomic DNA regions by hybridizing the targeted genomic DNA samples (fragmented either mechanically or enzymatically) with the generated hybridization probes, and eluting the captured genomic fragments by using conditions for releasing and separating the bound DNA from the hybridization probes.

1 . A method for selectively capturing and/or amplifying exons or targeted genomic regions from biological samples, comprising the steps of:
 (a) obtaining DNA templates for the targeted genomic regions;   (b) cloning the DNA templates into cloning vectors to form template DNA clones;   (c) constructing libraries of the template DNA clones that cover at least the targeted genomic regions;   (d) generating hybridization probes from the DNA template clones in the libraries;   (e) capturing the targeted genomic DNA regions by hybridizing with the targeted genomic DNA fragments with the generated hybridization probes; and   (f) eluting the captured genomic regions by using conditions for releasing the bound DNA from the hybridization probes.   
   
   
       2 . The method of  claim 1 , wherein the DNA templates are obtained by reverse transcriptions from total RNA or mRNA from the biological sample. 
   
   
       3 . The method of  claim 1 , wherein the DNA templates are obtained by performing multiplex polymerase chain reactions (PCRs), or by gene synthesis. 
   
   
       4 . The method of  claim 1 , wherein the DNA templates are generated for predetermined segments of mitochondria DNA, or the entire mitochondria DNA. 
   
   
       5 . The method of  claim 1 , wherein the cloning step comprises the step of ligating the DNA templates into a cloning vector or plasmid. 
   
   
       6 . The method of  claim 1 , wherein depending on the starting RNA materials, the libraries for the clones of the template DNA probes represent full-length RNA, open-reading frame RNA, or partial-length cDNA of the genes expressed by the biological samples. 
   
   
       7 . The method of  claim 1 , wherein the libraries of the template DNA clones represent the intron regions of interest, when the intron regions are amplified by multiple PCRs or by gene synthesis. 
   
   
       8 . The method of  claim 1 , wherein the template DNA clones in the libraries made from cDNA reverse transcriptions or PCR reactions are used to capture exons of the biological samples, wherein the template DNA clones in the libraries made from multiplex PCRs amplifying intron regions are used to capture intron regions of the biological samples, and wherein the template DNA clones in the libraries made from mitochondria DNA are used to capture mitochondria DNA of the biological samples. 
   
   
       9 . The method of  claim 1 , wherein the template DNA clones in the libraries are organizeable in a format that is manipulable by a robotic system. 
   
   
       10 . The method of  claim 1 , wherein the information of the template DNA clones is stored in a computerized database, the information including at least identity, sequence conformation, manufacturing date and location of each clone. 
   
   
       11 . The method of  claim 1 , where the constructing step comprises the steps of:
 (a) examining the quality and the completeness of the template DNA clones in the libraries; and   (b) monitoring and maintaining the quality of the template DNA clones in the libraries for long-term uses.   
   
   
       12 . The method of  claim 11 , wherein the examining step comprises the step of:
 (a) confirming the DNA sequence of each clone in the libraries; and   (b) comparing the DNA sequences of the clones with the reference DNA sequences of targeted genes or targeted genomic regions of the biological sample so as to check the completeness of the clones in the libraries.   
   
   
       13 . The method of  claim 1 , wherein the hybridization probes are generated by releasing DNA fragments hosted in the cloning vectors or plasmids with the use of restriction enzymes to digest the template DNA fragments/probes out of the cloning vectors or plasmids. 
   
   
       14 . The method of  claim 1 , wherein the hybridization probes are generated by PCR amplifications using the common primer pair sequences contained in the cloning vectors or plasmids in multiple cloning sites. 
   
   
       15 . The method of  claim 1 , wherein the hybridization probes are generated by using the cloning vectors or plasmids directly, without enzymatically cutting the hybridization probes out or PCR amplify the DNA fragments. 
   
   
       16 . The method of  claim 1 , wherein the hybridization probes are generated by in vitro transcriptions from DNA template clones. 
   
   
       17 . The method of  claim 1 , wherein the hybridization probes are generated by obtaining cDNA or cRNA of the genes by using in vitro reverse transcriptions of the templates DNA in the libraries. 
   
   
       18 . The method of  claim 1 , wherein the capturing step comprises the step of fixing the hybridization probes on surfaces of a solid supporting material or mixing the hybridization probes in a solution. 
   
   
       19 . The method of  claim 18 , wherein the conditions include changes of temperature, changes of salt solution, or changes of pH of solutions. 
   
   
       20 . A method for selectively capturing and/or amplifying targeted genomic regions from a biological sample, comprising the steps of:
 (a) providing libraries of template DNA clones that cover at least the targeted genomic regions;   (b) generating hybridization probes from the DNA template clones in the libraries; and   (c) capturing the targeted genomic DNA regions by hybridizing the targeted genomic DNA regions with the generated hybridization probes.   
   
   
       21 . The method of  claim 20 , wherein the providing step comprises the steps of:
 (a) obtaining DNA templates for the targeted genomic regions;   (b) cloning the DNA templates into cloning vectors to form template DNA clones;   (c) constructing libraries of the template DNA clones that cover at least the targeted genomic regions; and   (d) examining the quality and the completeness of the template DNA clones in the libraries.   
   
   
       22 . The method of  claim 20 , wherein further comprising the step of:
 (a) eluting the captured genomic regions by using conditions for releasing the bound DNA from the hybridization probes.   
   
   
       23 . The method of  claim 20 , wherein the targeted genomic regions comprises exons, subsets of exons, or desired regions of genomic, mitochondria and other forms of DNA from the biological sample including animalia, plantae, fungi, protista, archaea and/or eubacteria. 
   
   
       24 . A kit for capturing and/or amplifying targeted genomic regions from a biological sample, comprising:
 (a) libraries of template DNA clones that cover at least the targeted genomic regions, wherein the template DNA clones are formed by cloning the DNA templates obtained from the targeted genomic regions into cloning vectors;   (b) hybridization probes generated from the DNA template clones in the libraries; and   (c) means for hybridizing the targeted genomic DNA regions with the generated hybridization probes so as to capture the targeted genomic DNA regions.   
   
   
       25 . The kit of  claims 24 , wherein the hybridizing means comprises:
 (a) a solid supporting material having one or more surfaces on which the hybridization probes are placed for hybridization of the hybridization probes and the targeted genomic DNA regions; or   (b) a solution with which the hybridization probes are mixed for hybridization of the hybridization probes and the targeted genomic DNA regions.   
   
   
       26 . The kit of  claim 24 , wherein the libraries of the template DNA clones are managed manually, or managed by a spreadsheet program, or stored in a computerized database. 
   
   
       27 . The kit of  claim 24 , further comprising means for eluting the captured genomic regions. 
   
   
       28 . The kit of  claim 27 , further comprising means for detecting the eluted genomic fragments/regions.